Hypertonicity (e.g. high NaCl) activates the esential osmoprotective transcription factor NFAT5 by increasing its abundance, nuclear localization and transactivating activity. It is activated by a network of signaling molecules, whose members we have continued to identify and characterize, as follows: c-Abl. High NaCl-induced phosphorylation of NFAT5 at tyrosine-143 was known to be an important factor in increasing its activity in cell culture. We now find that NFAT5 also is phosphorylated at tyrosine-143 in rat renal inner medulla, dependent on the interstitial osmolality. c-Abl seemed likely to be the kinase that phosphorylates NFAT5 because Y143 is in a consensus c-Abl phosphorylation site. We now confirm that, as follows. High NaCl increases c-Abl activity. Specific inhibition of c-Abl by imatinib, siRNA, or c-Abl kinase dead drastically reduces high NaCl-induced NFAT5 activity by reducing its nuclear location and transactivating activity. c-Abl associates with NFAT5 (coimmunoprecipitation)and phosphorylates NFAT5-Y143 both in cell and in vitro. High NaCl-induced activation of ataxia telangiectasia mutated, previously known to contribute to activation of NFAT5, depends on c-Abl activity. Thus, c-Abl is the kinase responsible for high NaCl-induced phosphorylation of NFAT5-Y143, which contributes to its increased activity. PLC-gamma1. Phospholipase C-gamma1 (PLC-gamma1)has a predicted binding site at NFAT5-Y143. We found that: (i) Activation of NFAT5 transcriptional activity by high NaCl is reduced in PLC-gamma1 null cells and in HEK293 cells in which PLC-gamma1 is knocked down by a specific siRNA. (ii) High NaCl increases phosphorylation of NFAT5 at Y143. (iii) Wild-type PLC-gamma1 coimmunoprecipitates with wild-type NFAT5 but not NFAT5-Y143A, and the coimmunoprecipitation is increased by high NaCl. (iv) PLC-gamma1 is part of the protein complex that associates with NFAT5 at its DNA binding site. (v) Knockdown of PLC-gamma1 or overexpression of a PLC-gamma1-SH3 deletion mutant reduces high NaCl-dependent NFAT5 transactivating activity. (vi) Nuclear localization of PLC-gamma1 is increased by high NaCl. (vii) High NaCl-induced nuclear localization of NFAT5 is reduced if cells lack PLC-gamma1, if PLC-gamma1 mutated in its SH2C domain is overexpressed, or if Y143 in NFAT5 is mutated to alanine. (viii) Expression of recombinant PLC-gamma1 restores nuclear localization of wild-type NFAT5 in PLC-gamma1 null cells but not of NFAT5-Y143A. (ix)The PLC-gamma1 phospholipase inhibitor U72133 inhibits nuclear localization NFAT5 but not the increase of its transactivating activity. Thus, when NaCl is elevated, NFAT5 becomes phosphorylated at Y143, resulting in binding of PLC-gamma1 to that site, which contributes to NFAT5 transcriptional activity, transactivating activity, and nuclear localization. Phosphatases;SHP-1. Phosphorylation of NFAT5 contributes to its activation. Several of the kinases that are involved were previously identified, but the phosphatases were not. We screened a genomewide human phosphatase siRNA library in human embryonic kidney (HEK)293 cells for effects on NFAT5 transcriptional activity. We found that siRNAs against 57 phosphatases significantly alter TonEBP/OREBP transcriptional activity during normotonicity (290 mosmol/kg) or hypertonicity (500 mosmol/kg, NaCl added)or both.Most siRNAs increase NFAT5 activity, implying that the targeted phosphatases normally reduce that activity. We further studied in detail SHP-1, whose knockdown by its specific siRNA increases NFAT5 transcriptional activity at 500 mosmol/kg. We confirmed that SHP-1 is inhibitory by overexpressing it, which reduces NFAT5 transcriptional activity at 500 mosmol/kg. SHP-1 dephosphorylates NFAT5 at a known regulatory site, Y143, both in vivo and in vitro. It inhibits NFAT5 by both reducing NFAT5 nuclear localization, which is Y143 dependent, and by lowering high NaCl-induced NFAT5 transactivating activity. SHP-1 coimmunoprecipitates with NFAT5 and vice versa, suggesting that they are physically associated in the cell. High NaCl inhibits the effect of SHP-1 on NFAT5 by increasing phosphorylation of SHP-1 on Ser591, which reduces its phosphatase activity and localization to the nucleus. Thus, NFAT5 is extensively regulated by phosphatases, including SHP-1, whose inhibition by high NaCl increases phosphorylation of NFAT5 at Y143, contributing to the nuclear localization and activation of NFAT5. By siRNA screening, we previously found that protein targeting to glycogen (PTG), a regulatory subunit of protein phosphatase1 (PP1), contributes to regulation of high NaCl-induced NFAT5 transcriptional activity. high NaCl-induced inhibition of PTG elevates NFAT5 activity by increasing NFAT5 transactivating activity, protein abundance, and nuclear localization. PTG acts via a catalytic subunit PP1&#947;. PTG associates physically with PP1&#947;, and NaCl reduces both this association and remaining PTG-associated PP1&#947; activity. High NaCl-induced phosphorylation of p38, ERK, and SHP-1 contributes to activation of NFAT5. Knockdown of PTG does not affect phosphorylation of p38 or ERK. However, PTG and PP1&#947; bind to SHP-1, and knockdown of either PTG or PP1&#947; increases high NaCl-induced phosphorylation of SHP-1-S591, which inhibits SHP-1. Mutation of SHP-1-S591 to alanine, which cannot be phosphorylated, increases inhibition of NFAT5 by SHP-1. Thus high NaCl reduces the stimulatory effect of PTG and PP1&#947;. GSK-3&#946;. siRNA-mediated knock-down of GSK-3&#946; increases NFAT5 transcriptional and transactivating activities without affecting high NaCl-induced nuclear localization of NFAT5 or NFAT5 protein expression. High NaCl increases phosphorylation of GSK-3&#946;-S9, which inhibits GSK-3&#946;. In GSK-3&#946;-null mouse embryonic fibroblasts transfection of GSK-3&#946;, in which serine 9 is mutated to alanine, so that it cannot be inhibited by phosphorylation at that site, inhibits high NaCl-induced NFAT5 transcriptional activity more than transfection of wild-type GSK-3&#946;. High NaCl-induced phosphorylation of GSK-3&#946;-S9 depends on PKA, PI3K, and AKT, but not p38&#945;. Overexpression of PKA catalytic subunit &#945; or of catalytically active AKT1 reduces inhibition of NFAT5 by GSK-3&#946;, but overexpression of p38&#945; together with its catalytically active upstream kinase, MKK6, does not. Thus, GSK-3&#946; normally inhibits NFAT5 by suppressing its transactivating activity. When activated by high NaCl, PKA, PI3K, and AKT1, but not p38&#945;, increase phosphorylation of GSK-3&#946;-S9, which red